Endosomes are a heterogeneous population of vacuoles through which macromolecules internalized by endocytosis pass en route to lysosomes. In addition to transport, many critical functions of the endocytic pathway occur in endosomes, including the dissociation of internalized ligand-receptor complexes, the sorting of endocytic vesicle membrane and contents, receptor recycling, and the penetration of important pathogens (viruses, toxins) into the cytosol. Many of these functions depend on the maintenance of a low internal pH in endosomes. As in lysosomes and acidic secretory organelles, endosome acidification is accomplished by an ATP-driven proton pump. While superficially similar to the lysosome/granule-type H+ ATPase, little is known about the structural, catalytic, or bioenergetic properties of the endosome H+ ATPase, or about its genetic and biochemical relationship to the H+ pumps of other acidic organelles. This proposal describes an interdisciplinary study of the mechanism and functions of endosome acidification. We will continue to characterize and compare the acidification of endosomes and lysosomes using crude fractions labeled selectively with a pH-sensitive fluorochrome, fluorecein-dextran. More detailed bioenergetic studies will now be possible, however, given our ability to prepare highly enriched fractions of endosomes and lysosomes by a novel technique, free flow electrophoresis. In addition, we will analyze the properties of endosomes and lysosomes from different complementation groups of acidification-defective CHO cell mutants, in order to define their molecular defects. We also plan to identify the endosome/lysosome H+ ATPases by subjecting purified fractions to photoaffinity labeling and by preparing mono- and polyclonal antibodies to the organelle membranes and affinity-labeled proteins. Anti-ATPase antibodies will be used to purify the enzyme and for immunocytochemical, functional, and biogenesis studies. Additional studies on intact cells will be used to define 1) whether the endosome H+ ATPase is derived by endocytosis from the plasma membrane and 2) the time course of endosome acidification. The latter experiments will use a water soluble, recombinant fragment of the Influenza virus hemagglutinin as an intracellular pH probe. At acid pH, the hemagglutinin undergoes a biochemically and immunologically identifiable conformational change which will allow study of both the kinetics of acidification and, importantly, the distribution and morphology of the endosomes in which acidification occurs.